Nitroxides, which are potent antioxidants, are proving to have broad utility in a number of disease processes and/or conditions that represent excessive oxidative stress. The fact that nitroxides exert activity over such a range of disease conditions speaks to the importance of free radical reactions in tissue. Likewise, it is becoming apparent that free radicals are important in normal molecular signaling pathways and related gene expression. Further, nitroxide application (lead compound is Tempol) in the diet of mice was shown to result in hundreds of altered urine metabolic products including numerous Tempol metabolites. Of particular interest was the presence of 2,8-dihydroxylquinoline, which is related to the gut microflora. This finding prompted a study to determine if Tempol treatment would alter the gut microflora profile in mice. Tempol was administrated to mice and found to significantly change the gut microflora composition, in particular the Firmicute/Bacteroidete ratio consistent with a lean phenotype. The change in this ratio resulted from Tempol-medicated reduction of Lactobacillus and bile salt hydrolase activity leading to an accumulation of tauro-beta- muricholic acid (T-beta-MCA). Elevated T-beta-MCA inhibits farnesoid (FXR) signaling thus impacting lipid and glucose metabolism, which may underlie the anti-obesity properties of Tempol. We are presently repeating these studies with respect to Tempol influencing the gut microflora composition in different mouse facilities. Preliminary results show that the gut microflora composition varies substantially among different facilities as does the weight lowering capacity of Tempol. This study is currently being replicated. Another series of studies revealed that mice fed a high fat feeding exhibited marked increases in body mass and plasma lipid levels. Dietary Tempol (administered with the high fat diet) decreased both parameters. In the high-fat-fed mice significant elevations in plasma lipid levels and the inflammatory markers IL-6, MCP-1, MPO, SAA were detected, along with an increase in leptin and a decrease in adiponectin. Tempol supplementation reversed these effects. When compared to HFD-fed mice, Tempol supplementation increased plaque collagen content, decreased lipid content, and increased macrophage numbers. From these studies we concluded that Tempol may therefore be of value in suppressing obesity, metabolic disorders, and increasing atherosclerotic plaque stability. Tempol has been shown to protect against free radical mediated DNA damage for ionizing radiation and hydrogen peroxide. Tempol was shown to protect against DNA damage mediated by the reverse transcriptase inhibitor NRTI and zidovudine in cardiac tissue. Lastly, tumor growth in mice was shown to result in systemic oxidative stress as evidenced by DNA damage in a variety of organs. Tumor-bearing animals on a Tempol-containing diet exhibited significantly less DNA damage to organs. We are currently exploring the use of nitroxides as protectors against oxidative that exhibit slow reduction rates to the hydroxylamine. We feel that such nitroxides may be more efficient protectors that the ones we have evaluated in the past.